The goal of this research is to understand the molecular mechanisms by which protein kinases mediate the cellular actions of calcium. The approach in these studies will be one combining protein chemistry, enzymology, and molecular and cellular biology. Two calcium-dependent protein kinases will be purified to chemical homogeneity from bovine brain: myosin light chain kinase (a calmodulin-dependent enzyme) and protein kinase c (a calcium-sensitive but calmodulin-independent kinase). The nucleotide triphosphate specificity of myosin light chain kinase and its regulation by calcium, calmodulin and cyclic nucleotide-dependent protein kinases will be defined. Protein kinase C will be studied in a manner analogous to myosin light chain kinase. The nucleotide triphosphate, peptide and protein substrate specificity will be determined. In addition, the physical and kinetic characteristics and amino acid compositions of both protein kinase C and myosin light chain kinase will be thoroughly investigated. A substrate of myosin light chain kinase, brain myosin light chain, will be purified and the amino acid sequence of the phosphorylation site determined. Synthesis of peptide anologs of the phosphorylated site will facilitate determination of the structural requirements for phosphorylation. Other studies will include identification of effector binding domains and functional domains of the kinases, and amino acid sequence studies of phosphorylation sites on the kinases. The information obtained as a result of this research will contribute to our knowledge of calcuim regulation of protein phosphorylation as a post-translational regulatory mechanism. The investigations of nucleotide and protein substrate specificity will be the basis for a more rational approach to hte synthesis of pharmocological agents which affect the calcium- and phosphorylation-controlled cellular events. Finally, a greater understanding of the general and unique features of the various phosphorylation pathways found in eukaryotic biological systems might provide new insight into disease processes and the mechanisms of drug, hormone, neurotransmitter and toxin action as well as provide a firm base for future clinical studies.